The consultants identified loneliness and mental health as the most common problems of helpline callers at both measurement points. The results confirm a stable level of stress and well-being during the pandemic in TES consultants. However, they also show a slight decrease in perceived job-related meaningfulness. Well-being of counselors should be watched closely, as they are an important part of the psychosocial healthcare system.Telephone emergency service (TES) consultants have been challenged even more since the beginning of the pandemic. How the COVID-19 situation and the associated increasing demand for TES services affect the well-being and stress of TES counselors has not been assessed so far. This longitudinal study examined mental well-being (WHO-5), perceived stress level (PSS-10), and experienced job-related meaningfulness (CERES) of TES counselors at two measurement points during the pandemic. From December 2020 to January 2021 (t1), N = 374 counselors were recruited within the Austrian nationwide organization “TelefonSeelsorge”. From those, N = 108 also participated one year later at t2. Neither well-being nor perceived stress differed significantly between t1 and t2. There was a decrease in job-related meaningfulness (from 5.46 at t1 to 5.34 at t2;  The COVID-19 pandemic and associated lockdown measures markedly impact most aspects of daily life, including economy, education, healthcare, mental health, and domestic violence ,3, especTelephone emergency services (TES) are an important part of the psychosocial healthcare system , providiWe previously assessed the mental well-being and perceived stress level of TES counselors, as well as the main topics of helpline callers at the end of the first year of the COVID-19 pandemic in Austria . In brieIt has been suggested that the increased demand for mental health services augments the risk for burnout among mental health practitioners during COVID-19 . MoreoveThe current study aims to examine the mental well-being, perceived stress level, and job-related meaningfulness of Austrian TES counselors. As the increased demand on helpline counselors  might be an additional stressor and affect their well-being, a further aim was to assess potential changes in the topics of helpline calls with the prolongation of the pandemic.RQ 1:Do well-being, stress-level, and job-related meaningfulness in Austrian counselors of the TES change with the prolongation of the COVID-19 pandemic?RQ 2:Do the topics thematized by Austrian TES helpline callers change with the ongoing COVID-19 pandemic?To expand previous research, the following two research questions (RQs) were examined in the current study:This quantitative longitudinal study comprised two online surveys conducted with the Research Electronic Data Capture (REDCap) platform  ,22. The Counselors’ participation was voluntary and they received no incentives. Counselors had to give electronic informed consent by agreeing to the data declaration to start the survey. The principles outlined by the Declaration of Helsinki were followed and the study was approved by the ethics committee of the University for Continuing Education Krems (Austria) with the following protocol code: EK GZ 35/2018-2021.In Austria, the first COVID-19 cases were reported in February 2020, which was followed by a COVID-19 lockdown from the middle of March 2020 until the end of April 2020. After the nationwide lockdown, daily confirmed COVID-19 cases remained at a low level until summer 2020. With a strong increase in daily confirmed COVD-19 cases and hospitalization rates in autumn/winter 2020 (second wave of COVID-19 infections in Austria), further lockdown measures were introduced from the middle of November 2020 until the beginning of February 2021. During this time, the first survey of the current study was carried out (t1). After the openings on 8 February 2021, a third wave of infections hit Austria. Between May and July 2021, daily confirmed COVID-19 cases declined, and the vaccination rates increased significantly, which enabled a series of easing of COVID-19 restrictions. In late summer 2021, the “Delta” variant spread and the fourth wave of infections hit Austria. In view of the increasing number of hospitalized patients, new measures were introduced that increasingly restricted various areas of public life  for unvaccinated people starting from the middle of November 2021. The steady increase in COVID-19 patients in intensive care units led to a new nationwide lockdown from 22 November 2021. At the same time, the government announced a general compulsory vaccination from 1 February 2022. The general lockdown ended in the middle of December, whereas the lockdown for unvaccinated people remained in place. In late December 2021, the “Omikron” variant spread and the fifth wave of infections emerged in Austria ,24. The In accordance with the first survey, sociodemographic characteristics were surveyed .Well-being was measured with the WHO-5 inventory ,26. It aThe perceived stress level was measured with the Perceived Stress Scale-10 (PSS-10), a reliable and valid measure of the subjective stress level experienced over the previous month . The PSSExperienced job-related meaningfulness was assessed with the Concern, Enthusiasm, Relevance, Efficacy, Satisfaction (CERES) scale . The CERCounselors of the TES were asked to rate the frequency of 15 different topics thematized by helpline callers on a six-point Likert scale from “1 = never” to “6 = always”. The surveyed 15 topics researched are explained in detail in our companion paper .Statistical analyses were conducted with the IBM SPSS  Statistics 26 software program.t-tests for independent samples were conducted to investigate potential differences in the sociodemographic characteristics between counselors who responded or not to the second survey (responders vs. non-responders). Further t-tests were conducted to analyze potential differences in outcome variables  between responders and non-responders. The Bonferroni-corrected significance was set to p < 0.0167 (p < 0.05/3 t-tests). Differences in the topics of helpline callers between responders and non-responders were also investigated by t-tests, with the significance level set to p < 0.003 (p < 0.05/15 t-tests).Chi-squared tests and To evaluate potential differences in mental well-being, perceived stress, and job-related meaningfulness between the two time points, repeated-measures analyses of variance (RM-ANOVAs) were performed. The rating of mental health indices were the dependent variables. The time point (t1 vs. t2) was the within-subject factor. There were two between-subject factors: “gender” and “age”. The Greenhouse–Geisser-corrected values are presented. Bonferroni corrections were applied for the pairwise post hoc tests.p < 0.003 (p < 0.05/15 t-tests).To evaluate potential differences in the topics thematized by helpline callers between the two time points, pairwise t-tests were performed, with the significance being set to p < 0.05 before Bonferroni correction.All statistical tests were performed two-tailed, and the significance level was set to p = 0.699. Responders were, on average, M = 7.79 (SD = 7.98) years in profession at t1, which also did not differ from non-responders ; T  = −0.897; p = 0.380. The average working time at the TES at t1 was M = 16.55 (SD = 14.51) hours per month in responders and M = 15.19 (SD = 14.03) in non-responders; T  = 0.840; p = 0.401. As summarized in In total, out of 374 Austrian counselors of the TES who participated in the first online survey, 108 participated in the second survey (response rate = 28.9%). At the time of the first survey, responders were, on average, M = 57.26 (SD = 11.83) years old and did not differ from non-responders ; T  = −0.387; p-values ≥ 0.053). Perceived job-related meaningfulness was slightly higher in responders  compared to non-responders ; T  = 2.508; p = 0.013. No differences in the reported frequency of different topics were reported at t1 between responders and non-responders .Well-being and perceived stress level did not differ between responders and non-responders at t1 . No effect of gender was observed . An effect of age was observed for the WHO-5 and CERES (p ≤ 0.01), showing higher mental well-being and perceived job-related meaningfulness with increasing age; however, the perceived stress level was not affected by age (p = 0.069). No interactions between time and age nor time and gender were observed on all investigated variables .The counselors’ well-being and stress level did not differ between t1 and t2 . The CERp-values ≥ 0.057). At both time points, the topics loneliness and mental health were the most frequent topics of TES callers.As depicted in The major aim of this study was to evaluate whether mental well-being, psychological distress, and experienced job-related meaningfulness in TES counselors changed with the prolongation of the COVID-19 pandemic in Austria. Results suggest that, while mental well-being and the perceived stress level in TES counselors remained unchanged at t2 vs. t1, their perceived job-related meaningfulness declined. Overall, it has to be noted that obtained values for CERES were still on a high level at t2 (5.34), being higher compared to Austrian nurses surveyed before the COVID-19 pandemic (4.97) . To our The second aim of this study was to elucidate whether there are changes in the topics thematized by callers of the TES during the fourth wave of the COVID-19 pandemic in Austria (t2) compared to the second wave of the COVID-19 pandemic (t1). While the frequency of the different topics raised by helpline callers at t2 remained almost the same compared to t1, the quantity of helpline calls increased . During both time points, the topics loneliness and mental health were reported to be most often thematized by helpline callers. As reported in our previous paper, both topics have gained importance during the COVID-19 pandemic as compared to pre-pandemic times . The preper se. A further shortcoming is that the topics thematized by helpline callers were not assessed by objective data, but rather counselors’ self-reports.When interpreting the results, the following limitations should be considered. First, the final sample size was rather small. Second, no reference values of mental health and job-related meaningfulness were available for the Austrian general population during the second survey. Moreover, a measurement point prior to the pandemic would have been needed to evaluate whether counselors’ well-being, stress level, and job-related meaningfulness were affected by this state of public health emergence The results confirm a stable level of stress and well-being during the pandemic in TES consultants. However, they also show a slight decrease in job-related meaningfulness. Well-being of counselors should be monitored closely to prevent emotional exhaustion and ensure optimal low-threshold crisis intervention during and in the aftermath of the pandemic.


Xanthomonas oryzae (Xoo), a bacterium that causes serious disease in rice. Our results show that the decline in the pyruvate cycle (the P cycle) was a feature for ZSM resistance in the metabolome of ZSM-resistant strain (Xoo-ZSM), which was further demonstrated as the expression level of genes involved in the P cycle and two enzyme activities were reduced. On the other hand, alanine was considered a crucial metabolite as it was significantly decreased in Xoo-ZSM. Exogenous alanine promoted the P cycle and enhanced the ZSM-mediated killing efficiency in Xoo-ZSM. Our study highlights that the depressed P cycle is a feature in Xoo-ZSM for the first time. Additionally, exogenous alanine is a candidate enhancer and can be applied with ZSM to improve the antibiotic-mediated killing efficiency in the control of infection caused by Xoo.Microbial antibiotic resistance has become a worldwide concern, as it weakens the efficiency of the control of pathogenic microbes in both the fields of medicine and plant protection. A better understanding of antibiotic resistance mechanisms is helpful for the development of efficient approaches to settle this issue. In the present study, GC-MS-based metabolomic analysis was applied to explore the mechanisms of Zhongshengmycin (ZSM) resistance in  Xanthomonas oryzae pv. oryzicola (Xoo) is a Gram-negative bacterium that infects rice and causes bacterial blight (BB), also called bacterial leaf blight (BLB). Foliar symptoms of BB usually become evident at the tillering stage as small, green water-soaked spots at the tips and margins of fully developed leaves. The spots expand along the veins, merge, and become chlorotic and then necrotic, forming opaque, white to grey-colored lesions that typically extend from the leaf tip down along the leaf veins and margins ) separated Xoo-S from Xoo-ZSM . The varhttp://www.metaboanalyst.ca, accessed on 30 December 2021) and four pathways were significantly enriched, with a p-value < 0.05. The specific impact factor and p-value for each pathway are shown in The DAMs were submitted to MetaboAnalyst , a recently identified cycle providing respiratory energy in bacteria . The sigAlanine was one of the top three decreased metabolites in Xoo-ZSM, and the significantly reduced alanine, aspartate and glutamate metabolism pathway motivated us to investigate the influence of alanine on ZMS resistance in Xoo. The ZSM-resistant strain was cultured under the stress of 6 μg/mL ZSM and with gradient dosages of alanine ranging from 0 to 40 mM. A minimum of 10 mM alanine was required to achieve a ~12% reduction in percent survival, while any more did not seem to significantly further decrease the percent survival A, indicaIn the present study, metabolomic characterizations of two Xoo strains with different sensitivities to zhongshengmycin (ZSM) were compared and analyzed. The results show that the decline in the P cycle was a feature in Xoo-ZSM. Moreover, exogenous alanine promoted the P cycle and reversed the antibiotic resistance in the ZSM-resistant strain. The detailed results are discussed as follows.Edwardsiella tarda LTB4-RCIP grew slower than ciprofloxacin-sensitive LTB4-S in the first 8 h, and showed a disrupted pyruvate cycle, decreased energy metabolism, and increased fatty acid biosynthesis [nqrA or nqrF in Vibrio alginolyticus increased bacterial resistance to amikacin, gentamicin, and kanamycin, but led to reduced growth in the exponential phase. On the other hand, the metabolomics analysis revealed the promoted L-alanine catabolism and inhibited L-alanine anabolism [p-value < 0.05. In addition, Z-score analysis showed that 19 DAMs were increased while 24 DAMs were decreased in Xoo-ZSM when compared with that in Xoo-S. Furthermore, the 43 DAMs were submitted to MetaboAnalyst for KEGG pathway analysis. Four mainly infected pathways were enriched, while three of them, i.e., alanine, aspartate and glutamate metabolism, pyruvate metabolism, and glycolysis/gluconeogenesis, were associated with the metabolic category, and the other, aminoacyl-tRNA biosynthesis, was associated with genetic information processing, indicating that the variation in metabolism may play an important role in the ZSM-resistance development in Xoo. These results are consistent with previous reports that metabolic fluctuation affects the development of microbial antibiotic resistance [Firstly, Xoo-S and Xoo-ZSM were used for the metabolomic analysis and experimental performance. The growth curve of the two strains showed that Xoo-ZSM grew with a similar trend to Xoo-S, except for a relatively lower biomass, when compared with Xoo-S after the exponential phase. Interestingly, the tradeoff in growth seems to be a common phenotype in antibiotic-resistant strains, which may be attributed to the altered metabolism state, as shown in the central carbon metabolism, amino acid metabolism, and energy metabolism. For example, ciprofloxacin-resistant ynthesis . The absnabolism . As the nabolism ,22,23. Osistance ,24,25,26Vibrio alginolyticus [V. alginolyticus was also associated with the reduction in the P cycle. Metabolites in the P cycle reprogramed the colistin-resistant metabolome to a colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the P cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase [Edwardsiella piscicida, with the features of lower membrane potential and ATP [Secondly, we noticed that among the KEGG-enriched metabolic pathways, alanine, aspartate and glutamate metabolism, pyruvate metabolism, and glycolysis/gluconeogenesis are associated with the pyruvate cycle (the P cycle), a novel metabolic pathway that was recently proposed to provide energy to bacteria and affect the antibiotic uptake ,26. Accoolyticus . Colistieductase . In addi and ATP . To assaVibrio alginolyticus, exogenous L-alanine reverts the reduced Na+-NQR complex and promotes gentamicin-mediated killing [Edwardsiella tarda EIB202, alanine metabolism connects to riboflavin metabolism, which provides the source for reactive oxygen species (ROS) production, and in turn, potentiates the killing of kanamycin-resistant bacteria [Finally, alanine was found as a crucial metabolite that significantly promotes the P cycle and reverses the ZSM resistance in Xoo. With the supplementary alanine in the culture broth, the P cycle in the ZSM-resistant strain was elevated and the bacterial survival percentages were decreased with the addition of exogenous alanine under ZSM stress. The effect of alanine on bacterial antibiotic resistance was consistent with the findings in several other microbes. In  killing . In Edwabacteria . Therefobacteria ,33,34.To sum up, our findings highlight that the variation in metabolism is a crucial reason for the development of ZSM resistance in Xoo, while the depression of the P cycle can be drawn as a metabolic feature in the ZSM-resistant Xoo strain. Moreover, exogenous alanine reverses the depression of the P cycle, and enhances the efficiency of antibiotic-mediated killing. Our works not only reveal/partially reveal the mechanism of ZSM resistance in Xoo but also provide a novel strategy for controlling the ZSM-resistant Xoo in the field of crop protection.Xanthomonas oryzae strains were used in this study, which were Zhongshengmycin (ZSM)-sensitive bacteria (Xoo-S) and its ZSM-resistant strain (Xoo-ZSM). Xoo-S strain was in our lab store, while Xoo-ZSM was obtained by continuous culture of Xoo-S under the stress of ZSM with the concentration of half of the MIC for 34 generations. In the antibiotic bactericidal assays, the ZSM-resistant strain refers to a Xoo whose MIC is 16-fold higher compared with the Xoo-S. PSA medium or broth  were used for bacterial culture.Two v/v) products of the two strains were transferred into the 2 mL PSA fermentation broth with the addition of ZSM at gradient concentrations, followed by an 18 h shaken culture at 30 °C and 600 rpm. The OD600 value was observed by the MultiskanSky microplate instrument, and the MIC of ZSM to the strain was determined.After being cultured on PSA medium for 3 days initially, Xoo-S and Xoo-ZSM were transferred to 5 mL PSA broth and incubated at 30 °C, 200 rpm for 24 h for seed culture. After that, 1% (v/v) products of the seed culture were transferred to 50 mL PSA broth, followed by the shaken culture at 30 °C, 200 rpm. The biomass was valued as the OD600 readings every 4 h. Each trial contained at least three biological repeats.The 1%  was added immediately to quench cells at 4 °C for 1 h to end cell metabolism. After that, the bacteria were collected and stored at −20 °C. Five biological replicates and two technical replicas for each strain were performed in this study to eliminate the background errors and the instrumental errors among the individual samples.Strains were cultured as mentioned above. After 32 h fermentation, the cultures were centrifuged at 4 °C, 12,000 rpm for 5 min. The bacterial cells were collected and washed with 0.9% normal saline 3 times. After washing, the bacterial solution ODg. The methanol was evaporated in a 37 °C vacuum centrifuge dryer. The dried samples were then mixed with 80 μL methoxide pyridine hydrochloride purchased from Sigma-Aldrich (20 mg/mL). After ultrasonic treatment, the reaction was carried out at 37 °C for 3 h, then 80 μL of N-methyl-N-trimethylsilyl trifluoroacetamide  was added followed by a 30 min reaction at 37 °C to obtain acidic protons. Finally, the samples were centrifuged for 5 min at 4 °C at 12,000× g and used for treatment on GC-MS. GC-MS data were detected by thermal science tracer DSQII.The samples for GC-MS analysis were prepared following the methods mentioned before . Brieflyp < 0.05). The metabolic pathway was enriched by MetaboAnalyst4.0.The data deposition and statistic analysis were performed following the methods in the predecessors ,36. The The ZSM-resistant strain was cultured as described above. The well-cultured bacterial cells were collected at 8000 rpm for 5 min, washed three times with 30 mL sterile saline, and then suspended in PSA broth for 18 h culture. During this culture phase, ZSM with/without alanine was added into the PSA broth to determine the influence of alanine on ZSM killing efficiency. The alanine dosages were set from 0 to 40 mM. After incubation, 100 μL of samples were serially diluted and plated  onto PSA agar plates. The plates were cultured at 30 °C for 60–70 h depending on bacteria species. To determine colony-forming units (CFU) per mL, only those dilutions yielding 20–200 colonies were enumerated. Percent survival was determined by dividing the CFU obtained from a treated sample by the CFU obtained from control.Xanthomonas oryzae was extracted according to the Biospin Total RNA Extraction Kit step. The concentration and purity of total RNA of Xanthomonas oryzae were measured by μDrop plate, and the cDNA of X. oryzae was obtained by One-Step gDNA Removal and cDNA Synthesis SuperMix Kit . qRT-PCR was carried out on a 96-well plate containing 20 μL of liquid per well, including 10 μL 2× Perfect start Green qPCR SuperMix, 0.4 μL 50× Passive Reference Dye optional, 6.8 μL nuclease-free water, 2 μL cDNA template, and 0.4 μL per pair of specific primers. The detailed information of the primers is listed in The strains were cultured according to the method described above, and the expression level of related genes in the pyruvate cycle was detected by quantitative real-time PCR (qRT-PCR). Briefly, the total RNA  of g for 10 min, and the supernatant was transferred to a new centrifuge tube. The BCA protein quantitative/concentration determination kit  was used to obtain the protein standard curve so that the concentration of the protein sample could be detected. The activities of KGDH and SDH were determined by KGDH and SDH detection kits , respectively.The cell strains and the appropriate amount of extract (the ratio of 5 million cells/1 mL extract) were mixed and crushed by ultrasonic oscillation. The cells were broken with 60% intensity sound waves at 4 °C for 6 min, followed by centrifugation at 12,000×


The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC  primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2.Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house–designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (CIMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a clear potential for a long-lasting global pandemic and incapacitation of health systems . Aside fSeveral PCR-based techniques for detecting viral RNA have been approved as early diagnostic tests for SARS-CoV-2 infection; however, swab samples have shown varied positivity rates in PCR tests of COVID-19 patients , 7. A faIn this study, we validated the diagnostic sensitivity and specificity of RT-qPCR–based assays that identify the three major genes of SARS-CoV-2 in patients with clinical and subclinical COVID-19. Furthermore, we explored the diagnostic specificity and sensitivity of RT-qPCR in the upper- and lower-respiratory-tract samples, self-collected saliva samples, and other samples collected from the patients. So far, no prospective studies have proven the specificity and sensitivity of the Centers for Disease Control and Prevention (CDC) or World Health Organization (WHO) recommended RT-qPCR primers and evaluated their accuracy in clinical practice. In this study, we determined the specificity and sensitivity of different RT-qPCR primer sets, including the CDC and WHO primers, to assess and compare their accuracy.All patients were admitted and treated at a single tertiary hospital . Between February 21, 2020 and May 11, 2020, we enrolled 12 confirmed COVID-19 patients. The median age of all the patients was 49.16 years , including seven men and five women. Two of these patients were asymptomatic and did not have any clinical signs or symptoms of COVID-19 throughout the study period. In addition to the confirmed patients, 107 healthy subjects were enrolled, and 128 SARS-CoV-2-negative samples were collected in this study (Table S2). The clinical data for the patients with COVID-19 are listed in Table S3.E gene primer set of the Kogene Kit, the WHO RdRp primer set, and the CDC N2 and N3 primer set showed cross-reactivity with the purified SARS-CoV Urbani strain. Whereas WHO RdRp primer showed a cross-reactivity with influenza A virus with higher Ct value. However, the CDC N1 and N2 primer sets had cross-reactivity with influenza A, influenza B, and influenza C virus with Ct values above 35. Moreover, the reliability of the CDC N1 primer set differed from that of the other primer sets when the Ct value was set above >35. These data suggested that RT-PCR with either the iNP primer set or the RdRp primer set (Kogene Kit) has better specificity for the SARS-CoV-2 virus. The results are summarized in Numerous respiratory-disease viruses and bacterial strains were assayed by RT-qPCRs with all the primers evaluated in this study. The P = 0.006); coinciding with the detection of viral RNA up to day 14 to 15 in nasopharyngeal samples but only up to 7 to 8 days in the oropharyngeal samples at a Ct cutoff of 35 (Ct-35). In sputum samples, we consistently detected viral RNA with low Ct values. In two patients, viral RNA was detectable on 34th and 64th day of sputum sample collection (data not shown).In total, 590 various clinical samples, including nasopharynx (17.14%), oropharynx (15.25%), sputum (17.96%), saliva (11.69%), urine (15.59%), stool (9.32%), serum/plasma, and whole blood (13.05%) samples were analyzed for the presence of the viral RNA of SARS-CoV-2, as summarized in Table S4. For the 12 patients, the viral load in the samples starting from the onset of symptoms to the recovery phase was analyzed by RT-qPCR. The earliest sample was collected 2 days prior to symptom onset, and the latest sample was taken on the 74th day post-recovery. Almost all patients’ swabs tested positive for up to 7 days after symptom onset. Viral shedding from nasopharyngeal samples was substantially higher than that from oropharyngeal samples during the early stage of symptoms, from day 0 to day 7  had sensitivity and specificity of 84.2%/100% and 94.8%/100% in sputum samples and 60.9%/100% and 60.9%/100% in nasopharyngeal samples, respectively  had the highest sensitivity  in the sputum samples as compared with all the other primers. Nevertheless, in the nasopharyngeal samples, the sensitivity of iNP at Ct-35 was 69.9%, and the sensitivity of the CDC primers targeting genes N1 (69.6%), N2 (65.2%), and N3 (69.6%) were comparable, as presented in N2 gene (90.5% specificity in sputum and 96.4% in nasopharynx samples), respectively. Hence, RT-qPCR with the iNP primers was superior to RT-qPCR involving the (target gene E) Kogene Kit primers  when considering sensitivity in both sputum and nasopharyngeal samples .In brief, RT-qPCR using the primers for either the iNP assay or the  samples . Our resTo determine changes with time, we further analyzed the specificity and sensitivity of the samples collected from the patients depending on the time interval from the date of admission to complete recovery. The samples at different time points from the date of admission, namely, at 0 to 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, and 5 or more weeks, were analyzed for specificity and sensitivity .t-35 and Ct-40. As expected, the sensitivity of iNP RT-qPCR was significantly higher than that of the primers targeting genes E and RdRp in the Kogene Kit when the Ct cutoff was 35. Meanwhile, the positivity rate of sputum samples was much higher than that of nasopharyngeal samples, as illustrated in t, the sensitivity of iNP RT-qPCR at Ct-35 was significantly higher than that of the Kogene Kit. On the other hand, when the Ct cutoff was 40, the sensitivity markedly increased for both target genes E and RdRp of the Kogene Kit. This finding may be due to nonspecific bands similar to those reported in another study  during the first week after symptom onset . Our results indicated that sensitivity was highest in sputum samples, followed by nasopharyngeal, saliva, and oropharyngeal samples, as illustrated in We analyzed and compared the specificity and sensitivity of iNP RT-qPCR among various samples  at CA few authors have reported diagnostic accuracy in confirmed COVID-19 patients along with serological and culture-based results –17; howe–Present day diagnostic methods are based on RT-qPCR or deep-sequencing technologies that require some replication of a viral RNA to ensure that a sufficient amount of the virus is collected for diagnosis . Under tA few studies have assessed self-collected saliva and other samples for diagnosing COVID-19; however, to date, the selection of clinical samples for accurate diagnosis has not been specified , 26. In One study suggested that the rate of SARS-CoV-2 positivity in sputum samples is significantly higher than that for throat swabs, and sputum samples may be of greater significance for diagnosing COVID-19 . HoweverOur study shows the analytical and diagnostic specificity and sensitivity of various RT-qPCR primers used in clinical practice. Sputum samples yielded the highest sensitivity of RT-qPCRs, followed by nasopharyngeal, saliva, and oropharyngeal samples in patient’s diagnosis with COVID-19. Moreover, we report evidence that the CDC and WHO primers require considerable improvement for more accurate detection of SARS-CoV-2.We conducted a prospective cohort study on confirmed COVID-19 patients from February 21, 2020 to May 11, 2020 at Chosun University Hospital, South Korea. The participants were confirmed to be SARS-CoV-2 positive by diagnostic methods such as RT-qPCR, cell culture and a >4-fold increase or seroconversion in terms of the SARS-CoV-2 antibody titer. Negative controls were obtained from healthy subjects with no clinical symptoms, no history of contact with confirmed COVID-19 patients, and no history of antibody detection of a SARS-CoV-2.Nasopharynx and oropharynx swabs, sputum, saliva, urine, stool, serum/plasma, and whole blood were collected, and 200 μL of each sample was used for RNA extraction. Sputum, saliva, stool, and urine samples were self-collected by the patients. Sputum, saliva, and stool samples were diluted in phosphate-buffered saline (PBS), mixed, and centrifuged, and the supernatant was subjected to RNA extraction. Nasopharynx and oropharynx swabs were collected by a physician directly to the commercial UTM kits containing 1 mL of a viral transport medium  and were employed for RNA extraction. The viral RNA was extracted by a fully automated instrument  with the Real-prep Viral DNA/RNA Kit .2 at 37°C. The swab samples were obtained by means of the UTM kit with 1 mL of the viral transport medium , or the samples collected into collection tubes were diluted with 1 mL of Dulbecco’s phosphate-buffered saline  and thereafter inoculated into a monolayer of the cultured Vero cells. The inoculated culture was examined daily for cytopathic effects, similar to the procedures used for SARS-CoV and MERS-CoV in other studies (Vero E6 cells were used for the culturing and identification of SARS-CoV-2. All cell culture work of SARS-CoV-2 virus was performed in a laboratory with Biosafety Level 3 (BL3) facility . The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and a 1× penicillin–streptomycin antibiotic solution  in an atmosphere containing 5% CO studies , 33.2SO4, and optical density at 450 nm (OD450) was measured. The cutoff values were determined by calculating the mean OD450 plus 3-fold standard deviation of the negative serum samples. Thus, the observed cutoffs for IgG, IgM, and total antibodies were 1.1, 0.5, and 0.7, respectively. When the OD of a patient sample was greater than the calculated cutoff, it was considered positive for SARS-CoV-2.Serological assays, including tests for IgG, IgM, and total antibody  titers, were performed using an indirect ELISA. Briefly, each well of 96-well ELISA microplates  was coated with 100 μL of 2 μg/mL plant recombinant SARS-CoV-2 nucleocapsid protein  in carbonate-bicarbonate buffer followed by overnight incubation at 4°C. The microplates were washed with PBS supplemented with 0.05% Tween 20 (PBS-T) and blocked 5% skim milk in PBS-T for 2 h at 37°C. After washing, the serum samples were diluted 100-fold with blocking buffer and incubated at 37°C for 2 h. The plates were rewashed, and a secondary antibody  was added, and the plate was incubated again at 37°C for 1 h. After washing, 50 μL of the 3,3′5,5′-tetramethylbenzidine substrate  was added at room temperature and incubated for 30 min in dark. The reaction was stopped with 25 μL of 1 M H2 and were fixed with 80% acetone the next day. Each patient’s serum was subjected to 2-fold serial dilutions starting from 1:16 and then reacted with SARS-CoV-2 viral antigens in a moist chamber for 30 min at 37°C. After washing, the slides were further incubated with a 1:400-diluted secondary antibody . Then, the slides were observed under a fluorescence microscope  after the addition of mounting solution  on the slides. An IgG antibody titer ≥1:32 was chosen as the cutoff for the indirect immunofluorescent assay (IFA) using the clinical samples from 15 healthy individuals , whereas for target genes E and RdRp, the Kogene Kit  were utilized, and the amplification was carried out according to the manufacturer’s specifications. For iNP target, RT-qPCR was performed in Exicycler 96 Real-Time Quantitative Thermal Block , and for Kogene kits CFX96 Touch Real-Time PCR Detection System  was used. RT-qPCR optimization with Roche master mix (LightCycler Multiplex RNA Virus Master) was conducted with a gradient RT-qPCR and the conditions we set for optimal results. Cycle threshold (Ct) values were set to ≤35 and ≤40 for the reference gene and were assumed to denote a positive result. Ct values >35 were assumed to indicate a negative result. To compare the specificity and sensitivity of these primer sets with those of other neutral primers, we chose the primers recommended by the CDC and WHO. All primer details are listed in Table S1.RT-qPCR was performed targeting the P values <0.05. The mean data were used to evaluate sensitivity and specificity via the area under the receiver operating characteristic (ROC) curve. All statistical analyses were performed using the MedCalc software   and percentage . Categorical variables were compared using either the chi-square test or Fischer’s exact test, when appropriate. Continuous variables were compared using the Mann–Whitney nonparametric test, when appropriate. Statistical significance was assumed at Belgium) .


The MFT and MBI score significantly increased in both groups after the intervention (p < 0.001), but the effect size was greater in the task-oriented training group than the table-top activity training group. No significant interaction with MVPT-V score was found between the group and time (p > 0.05), and no statistical group difference was found either (p > 0.05). Both groups showed significant improvement in their MVPT-V score after the intervention (p < 0.001). The pilot study findings demonstrate that both task-oriented training and table-top activity training are effective in recovering upper-limb function, visual perception, and ADL in acute stroke patients.This pilot study aimed to investigate the effects of task-oriented training on upper-limb functioning, visual perception, and activities of daily living (ADL) in acute stroke patients. Of 20 participants, 10 were randomly assigned in a 1:1 ratio to experimental and control groups. Task-oriented training and table-top activity training were implemented for 6 weeks. Change in upper-limb functioning was assessed with the Manual Function Test (MFT); visual perceptual skill and ADL performance were evaluated using the Motor-Free Visual Perception Test—Vertical (MVPT-V) and Korean Modified Barthel Index (K-MBI), respectively. There was a significant interaction in the MFT and MBI score between the group and time ( In 2018, stroke was the fourth leading cause of death in Korea . It is dVisual perception is a process in which the central nervous system integrates visual information and helps withdecision-makingg by converting information arriving at the retina to cognitive concepts so that individuals can adapt to the environment . It aidsIn general, upper-limb dysfunction not only causes problems in performing tasks requiring fine motor skills  but also makes it very difficult to perform tasks requiring gross motor skills  . Upper-lIn rehabilitation, a goal of therapeutic training is to help patients try to use the affected side and voluntarily perform motor functions, and there are a variety of physical intervention approaches . Of thosAs discussed above, much research has been performed to investigate the effect of task-oriented training in stroke patients ,30,31,32The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Gachon University Institutional Review Board (1044396-202102-HR-034-01). All participants signed a statement regarding informed consent before beginning the study.A total of 27 acute stroke patients voluntarily participated in the study, and 3 patients who did not meet the inclusion criteria were excluded. The inclusion criteria of the study participants were: patients diagnosed with a stroke within three months based on CT and MRI findings. The following types of patients were excluded: patients with cognitive impairment or dementia with a Korean version of Mini–Mental State Examination (K-MMSE) score of 20 or lower who were unable to understand instructions; patients with a score of 4 or higher on the National Institutes of Health Stroke Scale (NIHSS); patients with orthopedic disorders; patients who could not be evaluated or treated because of the inability to understand oral instruction due to language disorders or other reasons; and patients with psychiatric disorders.n = 12) or a table-top activity training group (n = 12) using simple randomization methods with a computer-generated randomized table of numbers created before data collection.A total of 24 patients were randomly assigned into either a task-oriented training group , Motor-Free Visual Perception Test—Vertical (MVPT-V), and Korean version of Modified Barthel Index (K-MBI) were measured. Afterward, both groups underwent intervention for 30 m a day, five times a week, for 6 weeks. The interventions were provided by two occupational therapists with more than 7 years of experience. Measurements of MFT, MVPT-V, and K-MBI were conducted after 6 weeks of intervention in both groups .The MFT is a test used to assess a change in upper-limb functioning due to stroke. It can measure a change over a short duration during neurological recovery and is widely used in evaluating upper-limb functions and motor skills. The upper-limb function test in stroke consists of eight items, of which four pertain to upper-limb exercises, two to handgrip, and two to finger manipulation. The maximum total score is 32 points. The test is performed on both sides to predict the level of functional recovery of the upper limb on the affected side. In this study, the Manual Function Score was used .The MVPT-V is a standardized test used to evaluate visual perceptual skills in children and adults. It does not involve motor skills and thus can also be used for assessing patients with impaired motor function. Additionally, the test takes a shorter time to administer than other trials . The MVPThe K-MBI, which translated from the MBI developed by Shat et al., (1989)  by JeongIBM SPSS 26.0 software  was used to analyze the data. Data were summarized as means and standard deviations (SDs) for continuous variables and frequencies and percentages for categorical variables. The Kolmogorov—Smirnov test was used to examine the assumption of normality of the continuous variables. Chi-squared test and independent t-test were performed to compare the general characteristics of participants at baseline. Repeated measures analysis of variance (RM ANOVA) was conducted to determine whether there was the group-by-time interaction effect of interest. When a significant interaction was found, an independent t-test was performed to compare the differences at pre- and postintervention between the two groups. A paired t-test was also conducted to compare outcome variables before and after the intervention in each group. Then, an effect size of Cohen’s d was used to identify how large the mean difference between before and after the intervention in each group. The level of statistical significance was set to α = 0.05.Of a total of 24 study participants, 4  dropped out due to hospital discharge or transfer to another hospital. Thus, the data for the remaining 20 participants were included in statistical analyses. The general characteristics of the participants are described in p < 0.001), but the main effect of the group was not significant (p = 0.251). There was no significant difference between the groups at baseline (p = 0.741), while a significant difference was found after the intervention between the groups (p = 0.019). The MFT score significantly increased from 56.6 ± 12.9 to 79.4 ± 11.6 (p < 0.001) in the task-oriented training group, and the MFT score in the table-top activity training program also significantly increased, from 58.4 ± 11.0 to 65.0 ± 13.4 (p < 0.001).p = 0.013). The main effect of the group was not significant (p = 0.657), and no significant differences between the groups were found before or after the intervention . The MBI score in the task-oriented training group significantly improved, from 55.9 ± 14.4 to 80.9 ± 12.3 (p < 0.001). Likewise, the score in the control group significantly increased, from 59.2 ± 14.5 to 72.5 ± 12.6 (p < 0.001).Similarly, a significant group-by-time interaction effect was found in the mean MBI score (p = 0.141), and no statistical group difference was found either (p = 0.735). Both groups showed significant improvement in the MVPT-V score after the intervention .Regarding the total pre- and post-intervention MVPT-V scores, no significant interaction was found between the group and time  than table-top activity training (d = 0.538) after 6 weeks of intervention. Brain activation pattern as assessed by functional MRI was presented sporadically regardless of the lesion site in the stroke patients. However, it was specifically identified in the contralateral primary motor area and perilesional area after task-oriented training for upper limbs [Previous research reported that the task-oriented training group showed a significantly greater improvement in an upper-limb function test than the biofeedback system training group . Howeverer limbs . This reVisual perceptual therapy influences the recovery of visual perception, ADL performance, and cognitive function in stroke patients . FurtherFurthermore, there is little evidence for the effects of intervention approaches in improving visual perception . RegardiThe K-MBI test used to evaluate ADL performance showed that the score significantly improved in both groups, but no significant group effect was found. In some ways, the study findings are not in line with previous study findings that ADL performance significantly improved in task-oriented training groups compared to the groups that received occupational therapy intervention or action observation therapy intervention ,54,55. IHowever, the current study has limitations that need to be addressed. A follow-up measurement was not conducted to investigate the persistence of intervention effects. Additionally, even though there were no significant differences in types of strokes and affected sides between the groups, these variables could affect motor, visual, or cognitive function so these variables should be considered for further study. In terms of task selection, tasks were not prioritized for each patient by considering his/her impairments. Instead, the selected tasks were determined irrespective of patient needs. Thus, the study was not based on patient-centered tasks. Additionally, since this pilot study sample size was relatively small, future studies should investigate the long-term effects of a task-oriented training program with a sufficiently large sample by improving upon the limitations.The pilot study findings demonstrate that both task-oriented training and table-top activity training improve upper-limb function, visual perception, and ADL in acute stroke patients. In particular, the task-oriented training showed a greater improvement of the upper-limb function, so it is recommended that task-oriented training be applied for upper-limb function in acute stroke patients. Further studies should involve administrating various task-oriented training programs in chronic stroke patients to investigate the effects of upper-limb functioning and visual perception on ADL performance.


This study was conducted to compare the outcomes of prosthetic ring versus autologous pericardial strip for the treatment of functional tricuspid regurgitation during left-sided valve surgery by minimally invasive approach.From January 2008 and July 2016, autologous pericardial strip (group P-TAP) was used in 109 patients, and prosthetic ring (group R-TAP) in 115 patients. The primary outcomes were long-term overall survival, development of patch degeneration, and significant tricuspid regurgitation recurrence. The second outcome was the assessment of right ventricular functional parameters.p = 0.99). Severe regurgitation was observed in 1.8% of cases only in the R-TAP group (p = 0.49). There were no reoperations. Late mortality was 3.7% and 5.4% . Freedom from death, all causes, were comparable among groups (log-rank p = 0.45). There were no statistically significant differences between two groups in TAPSE, left ventricular end-diastolic diameter, left ventricular ejection fraction, and left atrial diameter.Operative mortality was 1 case (0.9%) in the R-TAP group. At the time of hospital discharge only one patient (0.9%) in the R-TAP group had grade III+ tricuspid regurgitation, and none had grade IV+. Mean follow-up was 94.1 ± 24.5 months. Mild and moderate tricuspid regurgitation recurrence was 3.7% and 4.5%  than annuloplasty with a prosthetic ring. In particular, the pericardial strip over time does not develop any degeneration or retraction. Secondary tricuspid regurgitation (TR) in the absence of organic lesion of the tricuspid leaflets is the most common form of tricuspid valve disease in developed countries. Over the past decade, a growing body of literature has addressed the criteria to perform concomitant tricuspid annuloplasty in patients undergoing surgery for left-sided heart valve disease . The degThe employment of a band of autologous pericardium to perform annuloplasty has been proposed for both the mitral and tricuspid valve , 7. One With the present investigation, we sought to compare the mild and long-term follow-up results of pericardial strip annuloplasty for secondary TR by minimally invasive surgery with those of state-of-the-art prosthetic ring annuloplasty. The purpose is to understand whether the different mechanical properties of these materials may influence the geometrical characteristics of the tricuspid valve and the right ventricle. Furthermore, in an historical period characterized by the increase in social costs and the reduction of hospital resources, it seems important to achieve state-of-the-art clinical results by containing the economic burdens associated with postoperative complications and the use of prosthetic material.Since 2008, all pre-, intra- and postoperative data of patients undergoing cardiac surgery at Anthea Hospital GVM Care & Research, Bari, Italy, are prospectively entered in an electronic database. Such dataset is managed by dedicated personnel and is periodically checked for completeness and consistency. The present report adopts the definitions and recommendations reported in the current guidelines for description of morbidity and mortality after heart valve operations . We querThe GVM Care & Research review board approved the study and need for patients’ consent was waived.The primary outcomes were long-term overall survival, development of patch degeneration or retraction, and significant tricuspid regurgitation recurrence. The second outcome was the assessment of right ventricular functional parameters in patients previously submitted to tricuspid valve annuloplasty for secondary TR using either a rigid prosthetic ring or an autologous pericardial band by minimally invasive approach.The indication to tricuspid annuloplasty was posed in compliance with the current guidelines . All opeThe employment of either technique for tricuspid annuloplasty was decided according to surgeon’s preference. In patients receiving P-TAP, a band of autologous pericardium  was harvested prior to establishment of CPB and treated with glutaraldehyde for 3 min. After exposure of the tricuspid valve, the strip was sutured to the tricuspid annulus with interrupted mattress sutures of 2-0 Ethibond suture  in correspondence of the anterior and the posterior leaflets. Two- to three-mm-interval sutures in the autologous pericardial strip and 5- to 6 mm-interval sutures in the tricuspid annulus can shorter the tricuspid annulus by 50 to 67%, as previously described . When tw®; Edwards Lifesciences) was sutured to the tricuspid annulus by standard operative techniques.In the case of R-TAP, a prosthetic semi-rigid incomplete ring  in all patients with the exception of a few operative mortality cases. All patients discharged alive from the hospital were followed up at our institution every year after the procedure. Visits included a physical examination with assessment of functional status, 12-lead electrocardiography, and trans-thoracic echocardiography. This was focused on the assessment and quantification of TR, left ventricular ejection fraction, end-diastolic diameters and left atrial diameter. The tricuspid valve regurgitation was classified into four grades: 0 for no regurgitation, I+ for mild regurgitation, II+ for moderate regurgitation, III+ for moderate-to-severe regurgitation, and IV+ for severe regurgitation. Recurrent TR at follow-up was defined as graded at least III+. At follow-up, the right ventricular function was quantitatively assessed by determination of the tricuspid annular plane systolic excursion (TAPSE) [The assumption of normality of each variable distribution was tested with the Shapiro–Wilk test. Normally distributed variables were reported as the mean ± standard deviation. Categorical variables are reported as number and percentage.t test and the chi-square test/Fisher’s exact test for continuous and categorical variables, respectively. The Kaplan–Meier method for survival analysis was employed, and the corresponding curves were built. All analyses were performed using R 2.13.2 software . The threshold for statistical significance was p < 0.05.The patients complying with the inclusion/exclusion criteria were divided into two groups according to the technique of tricuspid repair (either R-TAP or P-TAP). Operative mortality was defined as death within the 30th postoperative day. Intergroup comparison was performed using the two-tailed Student’s p = 0.84, 102.3 ± 9.06 and 104.6 ± 9.3, p = 0.16, in the P-TAP and R-TAP groups, respectively, Table p = 0.5.A total of 109 patients who received P-TAP and 115 patients who received R-TAP met the inclusion/exclusion criteria. The baseline characteristics of the study groups are summarized in Table 30-day mortality (defined as death within the 30th postoperative day) was 0.9% in the R-TAP group (1 patient died of cardiogenic shock), while no operative deaths were observed in the P-TAP group.p = 0.62). In the R-TAP group one patient died of cardiogenic shock, one for septic shock, and one for gastrointestinal complication. In the P-TAP the patient died of cardiogenic shock. The majority of patients who were discharged alive from the hospital presented TR of grade I+; 1 patient (0.9%) in the R-TAP group and none in the P-TAP group presented TR of grade III+, while no patient leaved the hospital with TR graded more than III+. Average hospital stay was 12 ± 7.6 days and 14.1 ± 10.8 days in the P-TAP group and R-TAP group, respectively  included three patients (2.6%) in the R-TAP group and one (0.9%) in the P-TAP group (p = 0.75). Among the ten mortalities, 4 were adjudicated to cardiac causes  and 6 were non-cardiac .Mean follow-up was 94.1 ± 24.5 months . Follow-up was conducted every year postoperatively in all patients; 4 cases in each group were lost at follow-up . Four (3.7%) and six (5.4%) patients died during the follow-up in the P-TAP and R-TAP groups, respectively (p = 0.99) and 2 case (1.8%) of recurrence III+/IV+ TR in the R-TAP group (p = 0.49); no patient received reoperation for TR. The patients with recurrent TR had preoperative low ejection fraction (38%), severe pulmonary hypertension, associated mitral regurgitation and dilated right ventricle.There were 4 (3.7%) and 5 (4.5%) cases of recurrence III+ TR in the P-TAP group versus R-TAP group (p = 0.54).Four patients were in NYHA III/IV in the P-TAP group versus 7 in the R-TAP group .Figure p = 0.71), left ventricular end-diastolic diameter , left ventricular ejection fraction , and left atrial diameter .Tricuspid valve repair resulted in near-normal morpho-functional parameters of the tricuspid valve (no patch degeneration or retraction) and right/left ventricle in both study groups. We observed no statistically significant differences between two groups at follow-up in terms of average TAPSE  than suture annuloplasty , 15. TheNonetheless, such study was limited by the comparison of this technique versus suboptimal approaches (suture annuloplasty only). To the best of our knowledge, this study is the first that directly compare the P-TAP technique for secondary TR versus the established prosthetic ring annuloplasty with a mild and long-term follow up in patients undergoing minimally invasive approach.The first major finding of the present investigation is the comparability of clinical and functional results of the P-TAP versus R-TAP technique in terms of long-term overall survival, development of patch degeneration or retraction, and significant TR recurrence during the long-term follow-up.We have shown that favourable clinical outcomes can therefore be obtained without the compelling need to use prosthetic materials for tricuspid annuloplasty. This is associated with lower rates of complications at late follow-up (lower risk of prosthetic endocarditis and thromboembolic events), and would determine a containment of hospital costs, since no dedicated consumables would be required. As far as the R-TAP method, the pericardial strip annuloplasty determined no events of atrioventricular block, it can be performed on the beating heart and adds little CPB time to the procedure.The second finding of our investigation is the comparability among the P-TAP and the R-TAP groups in terms of right ventricular functional parameters (TAPSE) at follow-up echocardiography. Right ventricular function is a major prognostic determinant of recurrence of TR in the long-term . Pericarp = 0.71), left ventricular end-diastolic diameter , left ventricular ejection fraction , and left atrial diameter .Preservation of such morpho-functional unit would be associated with reduced odds of cardiovascular mortality in the long-term follow-up . Long-teIn conclusion, the present study indicates the non-inferiority of the P-TAP technique for tricuspid annuloplasty (based on the use of a strip of autologous pericardium) in patients undergoing minimally invasive approach compared to the current gold-standard annuloplasty with the use of a prosthetic ring.Performing restrictive tricuspid annuloplasty using an autologous pericardial strip is associated to similar mild and long-term results, follow-up survival, rate of late tricuspid regurgitation and functional echocardiographic parameters than annuloplasty with a prosthetic ring. In particular, the pericardial strip over time does not develop any degeneration or retraction.This study has several limitations to be noted. First, this study was a retrospective observational study, non-randomized, performed at a single center. Second, the mean follow-up duration of 94 months is sufficiently longer but, given that late TR recurrence tipically matters 10 years after operation, further follow up might be required . Moreove


Metal–Organic Frameworks (MOFs) have the potential to change the landscape of molecular separations in chemical processes owing to their ability of selectively binding molecules. Their molecular sorting properties generally rely on the micro- and meso-pore structure, as well as on the presence of coordinatively unsaturated sites that interact with the different chemical species present in the feed. In this work, we show a first-of-its-kind tomographic imaging of the crystal morphology of a metal–organic framework by means of transmission X-ray microscopy (TXM), including a detailed data reconstruction and processing approach. Corroboration with Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) images shows the potential of this strategy for further (non-destructively) assessing the inner architecture of MOF crystals. By doing this, we have unraveled the presence of large voids in the internal structure of a MIL-47(V) crystal, which are typically thought of as rather homogeneous lattices. This challenges the established opinion that hydrothermal syntheses yield relatively defect-free material and sheds further light on the internal morphology of crystals. TXM-tomography unraveled large macropore defects within a MIL-47(V) MOF crystal. These pores do not seem to be well connected and they show a preferential orientation. COx, NOx or SOx, or the removal of contaminants from water.5,9–12 The high performance in such applications arises from their extremely high surface areas (often >1000 m2 g−1), regular pore systems that allow for (in principle) homogeneous diffusion, and the presence of interacting metal sites that may undergo reversible redox processes.13–17 This combination renders MOFs as materials of choice for advanced separations,18 being particularly interesting for mixed-matrix membranes.19–21Metal–Organic Frameworks (MOFs) have become a very important group of multifunctional porous materials, showing the first commercial applications after years of intense fundamental and more applied research.2,22–25 H2,26,27 alkanes,28–31 alkenes,32 aromatic hydrocarbons33–38 within the pores of MIL-47(V) have been previously published. These works highlight that separation and sorption properties of MOFs heavily rely on weak and strong interactions between the adsorbates and the internal micropore surfaces, i.e. organic ligands and metal sites, of the crystal lattice. Thus, the formation of defects or hollow cavities at the meso- (i.e. pore sizes > 50 nm),39 which may happen along with the deposition of nanosized deposits of impurities  disrupting the 1D channels of the structure may lead to alternative diffusion pathways, thus altering the separation properties.39 Therefore, understanding of the meso- and macropore structure in MOFs is crucial for developing refined synthetic methods.More specifically, many studies concerning diffusion and surface interactions of CO40 and are restricted, to the best of our knowledge, to a high-resolution transmission electron microscopy (HR-TEM) study of defect mesopores in UiO-66 crystals.41 Moreover, this technique remains restricted to volumes in the range of nm3, which prevents the user from probing large structures, e.g. a whole single-crystal. Other than that, fluorescence-lifetime imaging (FLIM) of UiO-66 tagged crystals has been recently reported, although rather than studying mesopore structure, the subject of matter was chemical heterogeneities.42 Several studies from multiple groups have shown how X-ray 3D imaging can be used for studying the porosity of porous materials at different length scales, overcoming the effects of severe beam damage and expanding the imaged volumes to the micron-range maintaining nanometer resolutions and allowing for tomography-based mass transfer simulations43–49 For instance, imaging of poisoning metal deposits within the pores of fluid catalytic cracking (FCC) particles with full-field transmission X-ray microscopy (TXM)43,50,51 and micro X-ray fluorescence (μ-XRF)43,52–54 allowed Kalirai and others to evaluate how the pores can become clogged during operation.55 Such studies require a 3D assessment and mapping of both deposits and the entire macropore space of individual catalyst particles with sizes in the 100 micrometre regime, that is, a method that provides both sufficient spatial resolution and a large field of view.Examples for imaging pores in MOFs are still scarce and rely mainly on electron microscopy, which often implies beam damage,et al., addressing issues such as spatially-resolved metal speciation and by-product phase formation56 However, this work focused on chemical heterogeneities in the crystal and not on porosity. Despite the tremendous importance of this property in the applications in which MOFs are typically used, to the best of our knowledge, no detailed studies focusing on porosity have been reported yet. Herein, we made use of full-field transmission X-ray microscopy (TXM) nanotomography to image a single MIL-47(V) crystal in 3D and map the macropore defects present within  could also cause such variation as the X-ray absorption coefficient (note that μ ∝ Z4), although the purity of the V precursor used was >99%. Hence, it seems unlikely the widespread presence of lighter or heavier elements throughout the crystal. To further validate these TXM-tomography results, six crystals were sectioned by using a focused Ga+ ion beam (FIB) and imaged by scanning electron microscopy (SEM). As shown in The data can therefore be used to qualitatively study differences in crystal defects even at resolutions smaller than the achieved spatial resolution of 230 nm. i.e. a given grayscale intensity value) must be selected. Every voxel with a grayscale intensity above this threshold will then be considered as solid while voxels below this value will be assigned as void space. As this crucial parameter can in principle be chosen arbitrarily, additional knowledge for selecting a correct value is needed. The threshold value determines the void voxel fraction of the reconstructed particle volume and is therefore directly correlated to the total porosity determined from the reconstructed data. To have an initial estimate for total porosity and in turn the threshold value, Hg-porosimetry characterization of the same material was performed resulting in a total porosity of 29% (see section 1). However, visual inspection of the grayscale cross sections  was used as a threshold value  bulk Hg-porosimetry evaluating many particles but suffering from uncertainties based on interparticle porosity; (ii) FIB-SEM providing high spatial resolution but being limited to studying a few crystal cross-sections; and (iii) 3D TXM data of a single but whole individual MIL-47(V) MOF crystal; we obtained a reasonable estimate for the macro-porosity of the sample. To further exploit the advantages of TXM and building upon the ability to evaluate the effect of different porosity values on pore connectivity and crystal accessibility, we continued our pore-network analysis using not only one but a series of grayscale threshold values corresponding to macro-porosities of 29%, 25%, 20%, 18%, 15%, 10%, 5%, and 2.6%. In the following section, we highlighted the boundary cases of 29, 18, and 2.6% porosity.Apart from the above-mentioned porosity overestimation, Hg-porosimetry has other limitations that lead to an underestimation of the pore volume. For example, closed pores cannot be probed as Hg cannot access them.3 along the primary MOF-crystal axis were computed  for molecular sorting.9,36,37 Hence, the findings presented up to here have two implications: they indicate that MIL-47(V) crystals synthesized by hydrothermal methods contain macroporous void regions  as crystallization defects. However, the connection between these macro-porous regions is not sufficient to create diffusion pathways that result in a significant loss of the MOFs sieving properties.To further evaluate connectivity, the different graphs (defined as a group of connected segment points), were analyzed separately using Matlab®. The 10 largest graphs obtained for each intensity threshold are plotted in i.e. connected pore sub-network). This vector points in the direction of the greatest spread of each graph (expressed as a cloud of points in space) and can therefore be used as a measure for pore orientation. Finally, in an attempt to elucidate the origin of the detected macropores, their orientation in relation to the MOF axis was assessed by calculating the first eigenvector (EV) of the covariance matrix of each graph (z-direction in the plot). These angles describe how parallel the graph orientation is with respect to the MOF axis (angle θ) and how the sub-network is oriented in the x–y plane (angle φ). θ (c) and φ (d) angles between the first EV of the graphs and the MOF axis. Based on these results there does not seem to be a clear preferred pore network directionality when inspecting the EVs that are projected perpendicularly to the MOF axis  MOF crystal with X-ray nanotomography. This method represents a non-destructive way of studying the internal pore space of MOF crystals and to characterize their internal architecture. In contrast to Hg-intrusion, TXM allowed to discriminate between porosity corresponding to internal voids, around 2–3% of the volume, and the interparticle space. The intrusion experiments clearly overestimated macro-porosity, rendering TXM a powerful alternative when detailed information of the internal macro-porosity is needed. Further numerical processing revealed that the observed macropores are not distributed homogeneously across the particle and their connectivity seems to be very poor. Also, as demonstrated by permeability simulations, they presumably only have a limited effect on mass transfer and therefore on the separation properties of the crystal for mixtures flowing through the MOF's internal pore space. By analysing the tilting angle of the pore regions, a preferred network orientation was observed. The pore sub-networks are mostly spread perpendicularly to the main MOF-axis, which could lead to anisotropies in diffusion and permeability. As the exact total porosity of the sample can only be estimated by cross-validating results from Hg-porosimetry, FIB-SEM, and X-ray nanotomography, it is not yet possible to quantify the exact extent of these effects.55,62–65 and highlights the added value of using X-ray microscopy as a non-destructive tool for MOF materials. This work is the first example of imaging of the inner macro-pore space of MOF materials at the single particle level and we predict that it will trigger further investigations of the internal porosity of other important topologies and morphologies.This is in line with studies from different fields that compare X-ray tomography with conventional pore analysis methods,et al.69 In short, vanadium metal powder , terephthalic acid , hydrofluoric acid , and DI water (molar ratio 1 : 0.25 : 2 : 250) were introduced in a Teflon-lined Parr steel autoclave for four days in an isothermal oven at 473 K . The green-yellow solid was collected by centrifugation and washed 3 times with DMF and ethanol (50 mL), then dried at 423 K under vacuum for 24 h. Powder X-Ray Diffraction (PXRD)  were outgassed for 24 h in air flow at 150 °C prior to loading them in the tube. Intrusion experiments were performed using an Hg-porosimeter Micromeritics Autopore IV 9510 in the pressure range p = 0.05 to 420 MPa. We used Washburn's equation (p = −4γ cos θ × d−1), with γ, mercury surface tension, and θ, contact angle, with values of 0.485 N m−1 and 130°, respectively, to calculate the pore diameter, d.MIL-47(V) crystals (+ ions were performed before imaging. SEM images of the cross section were recorded in backscatter electron (BSE) mode  and in secondary electron mode .Prior to measurements, the sample was coated with a Pt/Pd layer (∼10 nm) with a Cressington HQ280 sputter coater. Measurements were performed with a FEI Helios Nanolab 600 FIB-SEM instrument. The sample was placed on an aluminum stub using a carbon sticker. A protective layer of Pt (∼3 μm) was deposited on top of the region of interest before performing the measurements. For the FIB experiments, a trench was made by milling perpendicularly to the surface, next to the Pt-deposited area. After milling the trench, a cleaning step with Ga66,67 The beam energy was calibrated by measuring at the V K-edge of a reference metal foil. X-ray nano tomography was conducted below and above the V K-edge energies (5460.0 and 5482.0 eV) five times per angle with an angular step size of 1 degree over a range of 180 degrees, enabling a high-quality reconstruction of the 3D structure of the MOF single-crystal with the TXM-Wizard software package.59 The total duration of the scan was 260 minutes. Data analysis, processing and sample preparation details are further described in the ESIFull-field transmission hard X-ray microscopy (TXM) was performed at beamline 6-2C of the Stanford Synchrotron Radiation Lightsource at the Synchrotron Linear Acceleration Center (SLAC) National Accelerator Laboratory. Details of the experimental setup can be found elsewhere.3. The effective 3D spatial resolution was estimated to 230 nm by Fourier Shell Correlation (FSC) analysis of the tomography data , mesopores (2–50 nm) and small macropores could not be resolved.39 Each slice of the obtained 3D image was segmented manually, to determine the total particle volume (including pores) using Avizo Fire© software. Further details can be found in the Results and discussion and the ESI sections.†Each stack of 2-D projection images was aligned manually to correct for motor jitter and sample movement. Later, the 3-D tomographic slices were reconstructed with an iterative algebraic reconstruction technique (iART). TXM tomography data was binned from a 32 nm isotropic voxel size to a voxel size of 64 × 64 × 64 nmR. M. G.  reconstructed and analyzed the TXM tomography in discussion with F. M. (UU) and R. V. (UU); R. M. G. (UU) together with M. R. T. (UU) drafted the manuscript. M. R. T. and K. W. B. (UU) prepared the materials and collected XRD, TXM and routine data. N. N. (UU) carried out the FIB-SEM imaging of the cross-sections of the large crystals. K. W. B., B. S. (UU), F. M. and B. M. W. contributed to the proposal for beamtime and K. W. B., J. N.-W. (SSRL) and B. S. contributed to collecting the data at SSRL beamline 6-2c. J. Y. (ICV-CSIC) provided valuable input and performed the Hg-porosimetry measurements. F. M. and B. M. W. (UU) supervised the research and helped in the preparation of the article.The authors declare no competing or financial interests.SC-012-D1SC00607J-s001


The effects of ZnMgO ETL on the performance of InP QLED were investigated. X-ray diffraction (XRD) analysis indicated that ZnMgO film has an amorphous structure, which is similar to zinc oxide (ZnO) film. Comparison of morphology between ZnO film and ZnMgO film demonstrated that Mg-doped ZnO film remains a high-quality surface (root mean square roughness: 0.86 nm) as smooth as ZnO film. The optical band gap and ultraviolet photoelectron spectroscopy (UPS) analysis revealed that the conduction band of ZnO shifts to a more matched position with InP quantum dot after Mg-doping, resulting in the decrease in turn-on voltage from 2.51 to 2.32 V. In addition, the ratio of irradiation recombination of QLED increases from 7% to 25% using ZnMgO ETL, which can be attributed to reduction in trap state by introducing Mg ions into ZnO lattices. As a result, ZnMgO is a promising material to enhance the performance of inverted InP QLED. This work suggests that ZnMgO has the potential to improve the performance of QLED, which consists of the ITO/ETL/InP QDs/TCTA/MoO In the past decade, quantum dot (QD) has been considered a promising next-generation light-emitting material due to its outstanding photo-electrical properties ,2,3 and 2 and an external quantum efficiency (EQE) of 13.6% were realized for the first time [Developing eco-friendly, low-toxic or non-toxic QD materials is key to solving the above problems . Indium rst time . Howeverrst time . In generst time ,30. BaseIn this work, we introduced the ZnMgO nanoparticles as the ETL in InP-based red QLED with an inverted device structure. The effects of Mg-doping on the component, phase, morphology, and energy level structures of ZnMgO film were investigated. The performance of ZnMgO-based InP red QLED was analyzed. We verified that introduction of ZnMgO ETL can still improve the performance of InP QLED devices with an inverted structure. In addition, we found that Mg-doping can broaden the band gap of ZnO, which is mainly attributed to the conduction band minimum (CBM) shift upward. The optimizing of band structure and reducing of traps and defects result in improvements in turn-on voltage and luminance properties.3) was purchased from Sigma Aldrich .The ITO glass substrates were purchased from China Southern Glass Holding Corp.  with sheet resistance around 17 Ω/sq. ZnO and ZnMgO nanoparticle solution was purchased from Guangdong Poly OptoElectronics Corp. , with a concentration of 20 mg/mL in ethanol. The indium phosphide quantum dots (InP QDs) were purchased from Suzhou Xingshuo Nanotech Corp. , with the concentration of 25 mg/mL in normal octane. The 4,4′,4′′-tris(carbazol-9-yl) triphenylamine (TCTA) was purchased from Jilin OLED Material Tech Corp. . The molybdenum oxide . The as-deposited ETL was annealed at 120 °C for 12 min. Then the QDs layer was deposited on the ETL spin-coating at 3000 rpm for 30 s and annealed at 120 °C for 10 min (thickness: ~34 nm). The TCTA, MoO3 and Al electrodes were prepared by thermal evaporation process in turn, whose thickness was 60 nm, 7 nm and 110 nm, respectively. All processes were conducted in the glove box for isolating water and oxygen.Clean indium tin oxide (ITO) substrate was irradiated by UV light for 10 min to improve surface wettability. The InP QLED was fabricated with an inverted structure, consisting of the ITO/ZnO or ZnMgO/InP QDs/TCTA/MoOThe thickness of ETL and QDs was measured by a step profiler . The photoluminescence (PL) spectra were determined using a QE Pro spectrometer . The phase of ZnO or ZnMgO ETL was analyzed by X-ray diffraction . The ratio and chemical state of elements were determined by X-ray photoelectron spectroscopy . The current density (J), voltage (V), and luminance (L) characteristics were measured using a Keithley  2400 source meter and a Konica Minolta  Chroma Meter CS-200. Film morphology was acquired by atomic force microscope  and a scanning electron microscope . Electroluminescence spectra were recorded using a photometer . Time-resolved photoluminescence (TRPL) spectra were recorded using C11367-11 . Absorption spectra were obtained by UV-2600 , and the optical band gap was further calculated using the absorption spectra. Valence band maximum was analyzed by ultraviolet photoelectron spectroscopy .3/2 in ZnMgO (1021.1 eV) is higher than that of Zn 2p3/2 in ZnO (1021.0 eV), revealing that the Zn-O-Mg bonds are formed in ZnMgO film [MgO film . The sucTo investigate the effects of Mg-doping on the film surface, an AFM measurement was carried out for the ZnO and ZnMgO films . Figure The effects of ZnMgO ETL on the PL property of InP QDs were studied a. As canIn addition, a measurement of TRPL spectra for ZnMgO/QDs and ZnO/QDs was conducted to quantitatively evaluate the effect of defects on luminescence. In Equation (1), the exponential items of Among the three decay lifetimes of exciton recombination process, the long lifetime  of ZnMgO and ZnO was determined using the absorption spectra [hν is the photon energy that is calculated by the wavelength, A is a constant, and the value of n is 1/2 for the direct bandgap semiconductor of ZnMgO. The horizontal intercept of 2(αhν) versus photon energy curves represents the gE. gE of ZnMgO (3.7 eV) is higher than that of ZnO (3.53 eV).The band structure of ZnMgO and ZnO was investigated to understand the effects of Mg-doping on the position of the valence band and conduction band.  spectra ,33. The  spectra ,32,33,34gE value, which are 4.02 eV and 3.84 eV, respectively. The HOMO and LUMO of InP QDs were also measured  is higher than that of ZnO/InP-based QLED (4.03 cd A−1). The ZnMgO/InP-based QLED also realizes more than 1.3 × 104 cd m−2 maximum brightness, whereas the maximum brightness of ZnO/InP-based QLED is only 1.0 × 104 cd m−2.To verify this, the J-V-L curves of InP QLEDs with different ETL were recorded e. The inAn environment-friendly inverted InP QLED was fabricated using ZnMgO as electron transport layer. Element analysis demonstrated that Mg was alloyed into the ZnO lattice. The amorphous Mg-doping ZnO film retains a high-quality surface (RMS: 0.86 nm) as smooth as that of ZnO film, resulting in fewer traps or defects in InP QLED, which helps to enhance the irradiation recombination of QLED. In addition, we revealed that Mg-doping can broaden the band gap of ZnO, which is mainly attributed to the conduction band minimum (CBM) shift upward through band structure analysis. The enhancement in CBM level can reduce the barrier between ETL and QDs, resulting in the decrease in turn-on voltage from 2.51 to 2.32 V (reducing 0.19 V), which is basically consistent with the offset (0.18 V) of CBM after Mg-doping. The more matched energy band and fewer defects enhance the luminance property and current efficiency using ZnMgO ETL. This work verifies that the introduction of ZnMgO ETL can improve the performance of InP QLED devices with an inverted structure and suggests that the Mg-doping strategy is an efficient route to directionally regulate ZnO conduction bands. It provides a theoretical understanding for the optimization mechanism of inverted QLED’s performance using ZnMgO as ETL.